Journal of Applied Research and Technology
www.jart.ccadet.unam.mx JournalofAppliedResearchandTechnology15(2017)122–131
Original
Synthesis and characterization of novel ␣-monomers of peptide nucleic acid
Anjali Gupta
DepartmentofChemistry,SchoolofBasicandAppliedSciences,GalgotiasUniversity,GreaterNoida,U.P.,India Received20October2016;accepted20January2017
Availableonline27March2017
Abstract
Thispaperreportsthesynthesisandcharacterizationoftwonovelmodified␣-monomersofpeptidenucleicacidcontainingthymineandcytosine nucleobases.Thesenovelmonomershavebeensynthesizedbytheincorporationofthiolmoietyinthesidechainofpeptidenucleicacidbackbone.
Thesenovel␣-monomershavebeenidentifiedwithdifferentspectroscopictechniques,notablyinfra-redspectroscopy,1Hand13Cnuclearmagnetic resonancespectroscopy,supportedbymassspectrometry.
©2017UniversidadNacionalAutónomadeMéxico,CentrodeCienciasAplicadasyDesarrolloTecnológico.Thisisanopenaccessarticleunder theCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: PNA;Thiol;Cytosine;Thymine;␣-Monomers
1. Introduction
Peptidenucleicacids(PNAs),introducedbyNielsenandco- workersin1991are DNA/RNAanalogues(Nielsen, Egholm, Berg, & Buchardt, 1991), whereby N-2-aminoethylglycine repeatingunitsreplacethe sugar-phosphatebackbone andthe polyamidechainislinkedtonucleobasescovalentlythrougha carboxymethylspacer.Theattachednucleobasesarenaturaland sothroughWatson-Crickbasepairingareabletobindtocom- plementaryDNA/RNA (Egholmetal., 1993).Since thePNA backbone doesnot containany chargedphosphategroup,the absenceofelectrostaticrepulsiongivesrisetoastrongerbind- ingbetweenPNA/DNAstrandsthanthatofDNA/DNAduplex.
Consequently,the thermalstabilityof PNA/DNAduplexesas comparedtonaturalDNA/DNAdoublehelixofthesamelength is higher.Moreover, unlikeDNA/DNA, PNA/DNA duplex is muchlessaffectedbymediumwithhighionicstrengthmedium.
ItwasrecentlyfoundinlieuoffindingrelationshipofPNAtothe originoflifethatPNA-likematerialsarepresentincyanobacteria (Banacketal.,2012).
E-mailaddress:[email protected]
PeerReviewundertheresponsibilityofUniversidadNacionalAutónomade México.
The two strands inPNA/DNA hybrid canbe fashionedin eitherparallelorantiparallelorientationbutthelatermodegrabs higher stabilitybecauseof theachiralbackboneof PNA.The stabilityofthePNA/DNAduplexesisalsofoundtobehighly sensitivetotheexistenceofasinglemismatchedbasepair.PNA probesareverysequence-selectiveandadvancedtoDNAprobes insingle-basemismatchrecognition.Asenzymesaresubstrate specific,therecognitionofneutralbackboneofPNAisnoteasy byeithernucleasesorproteases,makingthempotentiallyresis- tanttoenzymaticdegradationandtheirstabilityoverwidepH range.
The majordrawbackslikepoor watersolubility,inefficient cellularuptake,self-aggregationandambiguityindirectionality ofbindingrestrictsitsapplicationswithinmedicine,diagnostics, molecularbiology,etc.Oneapproachtoimprovecellularuptake istoconjugatePNAstoawidevarietyofligands,suchasarti- ficial nucleases,peptides,intercalators or fluorescentreporter groups inordertocombine the favourableproperties ofboth entities inasingleconstruct.In mostoftheseconjugates,the attachment of ligandis either tothe C-or N-terminalend of thePNA.Overtheyears,anumberofbackbone-modifiedPNAs havebeenpreparedtostudytheeffectofintroducingchirality, chargeorstericbulkontheirpropertiessuchashybridizationor solubility. The application of a suitably protectedthiol mod- ified PNA monomer would give access to a PNA oligomer
http://dx.doi.org/10.1016/j.jart.2017.01.006
1665-6423/©2017UniversidadNacionalAutónomadeMéxico,CentrodeCienciasAplicadasyDesarrolloTecnológico.Thisisanopenaccessarticleunderthe CCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
containingasulfhydryl groupsuitable forpost-assemblycon- jugationmethods(DeKoningetal.,2002;DeKoning,VanDer Marel,&Overhand,2003;DeKoning,Filippov,VanderMarel, VanBoom,&Overhand, 2003;DeKoning,Filippov,Vander Marel,VanBoom,&Overhand,2004;DeKoningetal.,2006;
Dose&Seitz,2005;Goodwin,Holland,Lay,&Raney,1998;
Liu&Balasubramanian,2000).
2. Experimental 2.1. Generalprocedure
Melting points were determined on a sulphuric acid bath and are uncorrected. The IR spectra were recorded on a Perkin Elmermodel 2000 FT-IR spectrophotometer by mak- ing KBr discs for solid samples and chloroform film for viscous samples. The 1H NMR and 13C NMR spectra were recordedonBrukerAvance300spectrometerandBruker-500 using TMS as internal standard. The chemical shift values are on δ scale and the coupling constant values (J) are in Hz.EImassspectrawererecordedonAgilent-6210ESI-TOF.
Analytical TLCs were performed on pre-coated Merck sil- ica gel 60F254 plates with fluorescence indicator; the spots weredetectedbyviewingunderUVlightor byiodinecham- ber.Column chromatographywascarried outusing silicagel (100–200 mesh). U.V. data was recorded on Shimadzu UV- 2501PCUV–vis spectrophotometer.Allotherchemicalsused werepurchasedeitherfromS.D.FineChemicals,Spectrochem, IndiaorAldrichChemicalCo.,USAandusedwithoutfurther purification.
2.2. SynthesisofS-p-methoxybenzylcysteinemethylester hydrochloride(2)(Richter,Marsters,&Gadek,1994)
A solution of 4-methoxybenzyl chloride (5.8g, 37mmol) in dichloromethane (250mL) was added dropwise to a solution of l-cysteine (5g, 37mmol) in trifluoro acetic acid/dichloromethane(45mL/250mL), overaperiodof1hat 0◦C.Thereactionmixturewasstirredfor2hatroomtempera- ture.Thenmethanol(150mL)andwater(500mL)wereadded, thelayerswereseparatedandtheorganiclayerwasre-extracted withwater(100mL).TheaqueouslayerwaswashedwithDCM, filteredandsolventswereremovedinvacuo.Theresiduewas dissolved inwater, brought to pH 6 with10% NaHCO3 and theproductwasrecrystallizedfrommethanol/water(3:1).The obtainedcompound(5g)wasdissolvedinanhydrousmethanol (100mL)andthionylchloride (6mL)wasaddeddropwiseat 0◦C. The reaction mixture was stirred for 3h at room tem- perature.Methanol was then evaporated andthe residue was washed with diethyl ether. It was obtained as white solid (80%); meltingpoint:80◦C; 1H NMR(CDCl3, 300MHz):δ 2.88–2.91(m, 1H,Ha),2.98–3.04 (m, 1H, Hb), 3.77 (s,2H, H-5),3.78,3.82 (2×s,6H,H-1 andH-10), 4.12(m, 1H, H- 3), 6.88(d, J=8.7Hz, 2H, 2×H-8), 7.28(d, J=8.7Hz, 2H, 2×H-7); 13C NMR(CDCl3,125MHz): δ 31.62(C-4), 35.74
(C-5),52.66(C-1),53.27(C-3),55.97(C-10),114.69(2×C- 8), 130.38 (C-6), 131.11 (2×C-7), 159.24(C-9) and 169.52 (C-2); IR (KBr)νmax: 3412.57 (NH2 str),2840.36, 2637.14, 1745.85(C=O),1610.29,1581.69,1510.70,1439.71,1324.76, 1304.90, 1247.67, 1204.88, 1176.08, 1150.57, 1106.61, 1059.79, 1034.70, 995.41, 930.54, 870.01, 830.93, 777.10, 684.89and617.09cm−1;UV(methanol)λmax:278nm;HRMS:
C12H18O3NSCl[M]+291.2902.
2.3. SynthesisofN4-benzyloxycarbonyl
cytosine-1-yl-aceticacid(3)(Dueholmetal.,1994)
Benzyloxycarbonylchloride(5.2mL,36mmol)wasadded dropwisetoasuspensionofcytosine(2g,18mmol)inanhy- drous pyridine (100mL) at 0◦C. The mixture was stirred overnightand,subsequently,thepyridinesuspensionwasevap- orated to dryness. Water (20mL) was added and pH was adjusted to 1 with aqueous 4M HCl. The resultant white precipitate was filtered off, washed with water and partially dried. The wet precipitate was boiled with absolute ethanol (50mL) for 10min, cooled to 10◦C, filtered, washed thor- oughly with ether toyield compound 6 in 85% yield.Ethyl bromoacetate (0.5mL, 4.1mmol) was then added to a sus- pensionofN4-benzyloxycarbonylcytosine(1g,4.1mmol)and K2CO3 (570mg,4.1mmol)dissolved indimethylformamide (10mL). The reaction mixture was stirred overnight atroom temperature.The solutionwasthenfiltered andevaporatedto dryness. Thesolidresidue wastreatedwithwater(4mL)and 4MHCl(0.2mL),stirredfor15minat0◦C,filteredandwashed withwater.Water(2.5mL)wasaddedtoN4-benzyloxycarbonyl cytosine-1-yl-acetic acid ethyl ester (800mg, 2.4mmol) and 2M NaOH (3mL) and stirred for 2h. The reaction mixture was cooled to 0◦C and filtered. The compound was precip- itated by the addition of 4M HCl (2mL). The compound wasobtainedbyrecrystallizationwithmethanolaswhitesolid (60%);meltingpoint:272◦C(Literaturevalue=266–274◦C);
1H NMR (CDCl3, 300MHz): δ 4.54 (s, 2H, H-14), 5.19 (s, 2H, H-9), 7.06 (d, J=6.8Hz, 1H, H-5), 7.35–7.40 (m, 5H,C6H5),8.00(d, J=6.6Hz,1H, H-4);13CNMR (CDCl3, 75MHz): δ 50.54 (C-14), 66.53 (C-9), 94.03 (C-5), 127.95 (2×C-11),128.17(C-13),128.49(2×C-12),135.96(C-10), 150.42 (C-4), 153.18, 155.03 (C-2 and C-8), 163.31, 169.37 (C-6 and C-15); IR (KBr) νmax: 3423.87 (NHCO), 3143.94, 2953.22,2524.06,1759.05(NHCO),1712.63(COOH),1678.72 (C=O),1629.46,1582.61,1517.58,1454.32,1416.24,1371.21, 1332.40,1248.15,1215.41,1069.28,1007.24,981.10,901.54, 802.38, 780.06, 738.75, 694.29, 634.79, and 607.86cm−1; UV (methanol) λmax: 298nm; HRMS: C14H13O5N3 [M]+ 303.6239.
2.4. Synthesisof
[(methoxymethylcarbamoyl)-methyl]-carbamicacid tert-butylester(4)
Hydroxybenzotriazole (7g, 45.7mmol) was added to a solution of boc-glycine(5g, 28.6mmol) in anhydrous
dimethyl formamide (50mL) followed by the addition of N,O-dimethylhydroxylaminehydrochloride(4.5g,45.7mmol), DIPEA(7.9mL,45.7mmol)andDIPC(9mL,57.1mmol).The reaction mixture was stirred overnight at room temperature.
The solution was poured into water (100mL) and then fil- tered. The compound was extracted with ethyl acetate (3×
40mL).Theorganiclayerwaswashedwith10%KHSO4,10%
NaHCO3,waterandfinallybrine.Theorganiclayerwasdried withNa2SO4,filteredandevaporatedtoasmall volume.The productwaspurifiedthroughcolumnchromatographyusingsil- icagel(100–200mesh)inethylacetate/petroleumether(1:1).
Compound4wasobtainedaswhitesolid(80%);meltingpoint:
100◦C;1HNMR(CDCl3,300MHz):δ1.46(s,9H,H-1),3.21 (s, 3H, H-6), 3.72 (s,3H, H-7), 4.09 (s, 2H, H-4) and5.28 (brs,1H,NH); 13CNMR (CDCl3,75MHz): δ28.31(3×C- 1),32.37(C-6),41.68(C-4),61.41(C-7),79.60(C-2),155.86 (C-3) and 170.18 (C-5). IR (KBr) νmax: 3333.33 (N-H str), 3289.71,3006.83,2975.75,2932.75,1717.43(COO),1658.26 (NCO),1619.57,1572.61,1447.43,1401.80,1365.72,1285.99, 1250.32, 1170.30, 1128.03,1030.63, 985.00,946.16, 871.52, 807.40,780.10,761.18and621.05cm−1;HRMS:C9H18O4N2
[M]+218.2502.
2.5. Synthesisof2-(bocamino-ethylamino)-3-(4- methoxybenzylsulfanyl)-propionicacidmethylester (6)
A solution of [(methoxymethylcarbamoyl)-methyl]- carbamic acid tert-butyl ester (3g, 15.9mmol) in anhydrous diethyl ether (25mL) was added dropwise in a suspension of LiAlH4 (726mg, 19.2mmol) in diethyl ether (25mL) at
−30◦C.Thereaction mixturewaskeptat0◦Covernightand then 10% KHSO4 was added to quench excess of LiALH4. Theorganic layerwaswashedwith10% NaHCO3andbrine.
The solution was dried on Na2SO4 and the solvent was then evaporated to yield crude aldehyde. The aldehyde (2g, 12.6mmol) was dissolved in anhydrous methanol (25mL) andtoit,methyl-2-amino-3-(4-methoxybenzylthio)propanoate hydrochloride (2, 3.7g, 12.6mmol) and NaCNBH3 (1.5g, 25.2mmol) were added. The reaction mixture was stirred overnightatroomtemperature. Methanolwasevaporatedand thentheresiduewasdissolvedinethylacetate.Theorganiclayer waswashedwith10%KHSO4,10%NaHCO3andfinallybrine.
The product was purified through column chromatography using silica gel (100–200 mesh) in ethyl acetate/petroleum ether(1:10).Compound6wasobtained asviscousoil(40%);
1H NMR(CDCl3,300MHz):δ 1.45 (s,9H,H-8), 2.58–2.63 (m,1H,Hb),2.67–3.16(m,3H,H-4,H-5andHa),3.30–3.39 (m, 1H, H-2), 3.51 (brs,1H, NH), 3.69 (s, 2H,H-10), 3.73, 3.79 (2×s, 6H, H-9 and H-11), 5.02 (brs, 1H, NH), 6.85 (d, J=8.4Hz, 2H, H-2) and 7.23 (d, J=8.4Hz, 2H, H-3);
13CNMR (CDCl3,75MHz):␦ 28.43(3×C-8), 34.25(C-3), 36.18 (C-10),47.27 (C-4), 52.11 (C-5), 55.29 (C-11), 60.21 (C-2), 60.55 (C-9), 79.17 (C-7), 113.94 (2×C-2), 129.83 (C-1), 130.00 (2×C-3),156.04 and 158.72 (C-4 andC-6),
174.03 (C-1). IR (KBr) νmax: 3357.90 (N-H str), 2976.13, 1735.99, 1708.90, 1610.44, 1512.65, 1459.59, 1390.88, 1366.35,1249.95,1173.22,1035.08,832.51and775.30cm−1. UV (methanol) λmax: 279nm. HRMS: C19H30O5N2S [M]+ 398.8868.
2.6. Generalprocedureforsynthesisof2-[{2-(Base-1-yl)- acetyl}boc-aminoethylamino]-3-(4-methoxybenzyl sulfanyl)-propionicacidmethylester(7and8)
2-(2-Bocamino-ethylamino)-3-(4-methoxybenzylsulfanyl)- propionic acid methyl ester (1g, 2.4mmol) was dissolved in anhydrous dimethyl formamide (15mL). At 0◦C, base-1-yl-acetic acid (3.6mmol) and 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) (700mg, 3.6mmol) were added. The reaction mixture was stirred overnight and then added to ice cold water (10mL).
Thecompoundwasextractedwithchloroformandtheorganic layerwaswashedwith10%NaHCO3andbrine.Theproduct wasobtainedthroughcolumnchromatographyusingsilicagel (100–200mesh)inethylacetate/petroleumether(3:2).
2.7. 2-[{2-(Thymin-1-yl)-acetyl}boc-aminoethylamino]-3- (4-methoxybenzylsulfanyl)-propionicacidmethylester (7)
Compound 7 was obtained as white solid (70%); melting point: 68–70◦C;1HNMR(two rotamers,CDCl3,300MHz):
δ 1.45 (s, 9H, H-9), 1.91 (s, 3H, H-12), 3.09–3.13 (m, 5H, H-6, H-7 andH-2), 3.69–3.79 (m, 10H, H-2, H-4, H-5 and H-15),4.45–4.61(m,2H,HaandHb),5.30(brs,1H,NHBoc), 6.85 (m,3H,H-2 andH-10), 7.22(d,J=8.1Hz,2H,H-3);
13CNMR(CDCl3,75MHz):δ12.35(C-12),28.40(3×C-9), 30.53(C-3),36.68(C-4),38.56(C-6),48.13(C-7),48.56(C-2), 52.85(C-5),55.31(C-15),61.06(C-2),80.00(C-8),110.67(C- 11),114.05(2×C-2),130.02(2×C-3,C-1),140.79(C-13), 150.73,156.87and158.85(C-4,C-14andC-16),163.98(C- 10),167.22and170.17(C-1andC-1);IR(KBr)νmax:3343.66 (N-Hstr),3201.56,3010.30,2838.28,1680.61(COO),1610.92, 1512.24, 1465.46, 1368.35, 1302.84, 1247.87, 1174.14, 1107.92,1032.54,965.46,834.78,755.41and667.00cm−1;UV (methanol)λmax:269nm;HRMS:C26H36O8N4S[M+H+Na]+ 587.9988.
2.8. 2-[{2-(N4-Cbz-cytosin-1-yl)-acetyl}boc-
aminoethylamino]-3-(4-methoxybenzylsulfanyl)-propionic acidmethylester(8)
Compound 8 was obtained as white solid (60%); melting point: 66–68◦C; 1HNMR (two rotamers, CDCl3,300MHz):
δ 1.47 (s, 9H, H-10), 3.02–3.38 (m, 5H, H-2, H-6 and H- 7),3.59–3.83(m,10H, H-2,H-4,H-11andH-5),4.59–4.88, (m, 2H, Ha and Hb), 5.26 (s, 3H, H-17), 5.54 (brs, 1H, NHBoc), 6.88 (d, J=8.1Hz, 2H, H-2), 7.26 (d, J=7.8Hz, 2H,H-3),7.31–7.51 (m, 7H,Ar-Cbz, H-12 andH-13);13C NMR (Acetone-d6, 75MHz): δ 28.62 (3×C-10), 31.16 (C- 3),36.33(C-4),39.82(C-6),49.24(C-7),50.85(C-2),52.57 (C-11), 55.54 (C-5), 61.49 (C-2), 67.83 (C-17), 79.29 (C- 9), 94.67 (C-13), 114.77 (2×C-2), 128.87, 129.04, 129.36 (C-19, C-20 and C-21), 131.15 (2×C-3), 132.44 (C-1), 137.08 (C-18), 151.35 (C-12), 153.91, 156.90, 158.15 and 159.78 (C-4, C-8, C-15 and C-16), 164.12, 168.39 and 170.90 (C-1, C-1 and C-14); IR (KBr) νmax: 3407.37 (N- Hstr),3015.79, 2850.36, 1744.75(COO), 1664.33, 1630.62, 1561.35, 1508.96, 1454.23, 1369.11, 1249.01, 1214.04, 1062.19,1034.00, 1002.03,755.25,698.11 and667.21cm−1. UV(methanol)λmax:302nm;HRMS:C33H41O9N5S[M+H]+ 684.1031.
2.9. Generalprocedureforsynthesisof2-[{2-(base-1-yl)- acetyl}boc-aminoethylamino]-3-(4-methoxybenzyl sulfanyl)-propionicacid(9and10)
2-[{2-(Base-1-yl)-acetyl}boc-aminoethylamino]-3-(4- methoxybenzylsulfanyl)-propionicacidmethyl ester(500mg) was dissolved in methanol (3mL) and 4M LiOH solution (2mL) was added to it. The reaction mixture was stirred overnight and then methanol was evaporated. The mixture was acidified to pH 5 by the addition of 2M HCl solu- tion. The precipitate was then filtered and washed with ether.
2.10. 2-[{2-(Thymin-1-yl)-acetyl}boc-aminoethylamino]- 3-(4-methoxybenzylsulfanyl)-propionicacid
(9)
Compound9wasobtainedassemisolid(60%);1HNMR(two rotamers,Acetone-d6,300MHz):δ1.42(s,9H,H-9),1.81(s, 3H,H-12),3.30–3.50(m, 5H,H-2,H-6,andH-7), 3.71–3.77 (m,7H,H-2,H-4andH-5),4.29(brs,1H,NH),4.66–4.83(m, 2H,Ha andHb),6.86(d,J=8.7Hz,2H,H-2)and7.25–7.29 (m,3H,2×H-3andH-10);13CNMR(Acetone-d6,75MHz):
δ13.35(C-12),29.70(3×C-9),31.24(C-3),36.36(C-4),39.76 (C-6),49.01(C-7),49.19(C-2),55.54(C-5),61.39(C-2),79.37 (C-8),109.83(C-11),114.78(2×C-2),114.85(C-1),131.04 (2×C-3),142.62(C-13),151.91,153.46and156.86 (C-4, C-14andC-15),159.77 (C-10),164.86and168.40 (C-1 and C-1); IR (KBr) νmax: 3422.33 (N-H and COOH), 2974.78, 2930.22, 1675.98, 1512.18, 1465.01, 1367.77, 1248.77, 1171.39, 1031.70, 964.96,835.12, 780.41, and552.03cm−1; UV(methanol)λmax:268nm;HRMS:C25H34O8N4S[M+Na]+ 573.4542.
2.11. 2-[{2-(N4-Cbzcytosin-1-yl)-acetyl}Boc-
aminoethylamino]-3-(4-methoxybenzylsulfanyl)-propionic acid(10)
Compound 10wasobtained aswhitesemisolid (55%); 1H NMR (two rotamers, CDCl3, 300MHz): δ 1.43 (s, 9H, H- 10),2.95–3.59(m,5H,H-2,H-6andH-7),3.64(s,2H,H-4), 3.72–3.79(m,5H,H-2andH-5),4.85–4.96(m,2H,HaandHb), 5.24(s,3H,H-16),6.21(brs,1H,NHBoc),6.87(d,J=8.7Hz, 2H,H-2),7.28(d,J=8.4Hz,2H,H-3),7.33–7.47(m,6H,Ar- Cbz,H-12),7.87(d,1H,J=7.2Hz,H-11);13CNMR(CDCl3, 75MHz):δ28.43(3×C-10),30.51(C-3),36.61(C-4),38.82 (C-6), 49.06 (C-7), 49.73 (C-2), 55.32 (C-5), 61.15 (C-2), 67.89 (C-16), 79.95 (C-9), 94.99 (C-12), 114.02 (2×C-2), 128.26–128.69(C-18– C-20),130.10 (2×C-3),131.53(C- 1),135.06(C-17),149.59(C-11),152.35,155.37and155.99 (C-4,C-8andC-14),158.78,162.91,167.09and170.20(C- 1, C-1, C-13 and C-15); IR (CHCl3) νmax: 3423.82 (N-H str), 2927.79, 2851.54, 1700.69 (COOH), 1686.74, 1655.94, 1511.48, 1457.34, 1367.83, 1250.34, 1211.18, 1174.83, 1032.25, 833.56, 774.82 and 609.79cm−1; UV (methanol) λmax: 310nm; HRMS: Calculated for C32H37O9N5S [M]+ 667.4619.
3. Resultsanddiscussion
Thesynthesisofthemonomerstartedwithboc-glycineand N,O-dimethylhydroxylamine,whichwasconvertedtoitsWein- rebamide,[(methoxymethylcarbamoyl)-methyl]-carbamicacid tert-butyl ester (4) inabout80% yield.In its IRspectrum,it showed characteristic peaks at 3333cm−1 that describe N-H stretching. In the 1H NMR spectrum (Fig. 1), a characteris- ticsingletatδ1.46fornineprotonsandcorresponding peaks in 13C NMR at δ 28.31, 79.60 and 155.86 confirm the tert- butyloxycarbonyl (boc)group.Twosinglets in1HNMR atδ 3.21andδ3.72eachintegratingforthreeprotonsweredueto methylandmethoxygroupsattachedtonitrogen,respectively.A
7.281 5.281 4.086 3.716 3.205 1.456 0.000
8 7 6 5 4 3 2 1 0 ppm
1
2 O O
O NH
N OCH3
3 4
5 6
7
10.14
3.363.38
2.28
1.00
Fig.1.1HNMRspectraof[(Methoxymethylcarbamoyl)-methyl]-carbamicacidtert-butylester(4).
singletcorrespondingtoglycinemethylenegroupwasobserved atδ4.09.
ReductionofWeinrebamidewithlithiumaluminiumhydride yieldsanaldehyde,(2-oxo-ethyl)-carbamicacidtert-butylester (5) and its subsequent reductive amination with methyl-2- amino-3-(4-methoxybenzylthio)propanoate hydrochloride (2) yieldsPNAbackbonethatcontainsligationhandle,sulfhydryl groupat␣-position.Compound(6)wasobtainedin40%yieldas colourlessoil.InitsIRspectrum,itshowedcharacteristicpeaks at3357cm−1thatdescribeN–Hstretching.Inthe1HNMRspec- trum(Fig.2),acharacteristicsingletat␦1.45fornineprotons wasobservedforthethreemethyl groupsof bocandthecor- respondingcarbonsin13CNMRappearedatδ28.43whilethe boctertiarycarbonappearedatδ79.17.Amultipletintherange δ 2.58–2.63was assigned tooneof thediastereotopic proton Hb.Anothermultipletintherangeδ2.67–3.16wasobservedfor anotherdiastereotopicprotonHaandtwomethylenegroups(H- 4andH-5)ofethylenediamineunit.Thecorrespondingcarbons forC-4andC-5appearedatδ47.27andδ52.11,respectivelyin
13CNMR.Thechiralcentreofcysteine,i.e.H-2appeareddown- fieldasmultipletintherangeδ3.30–3.39becauseoftheattached thiollinkage.Asingletformethylenegroupattachedwiththe sulfhydrylgroupwasobservedat␦3.69,whiletwosingletscor- respondingtotwomethoxygroupswereobservedatδ3.73and δ3.79thatexplaintheincorporationofsuitablyprotectedcys- teineinthebackboneat␣-position.Twoorthocoupleddoublets (twoprotonseach)atδ6.85(J=8.4Hz)andδ7.23(J=8.4Hz) wereassignedtothearomaticprotonsofS-benzylgroup.In13C NMRspectrum,peaksatδ 34.25andδ 36.18wereidentified fortwo methylenegroups,C-3andC-10attachedtothethiol group.Thetwomethoxygroupswereobservedatδ55.29and δ60.55,whilethecarbonyloftheestergroupwasobservedatδ 174.03.
Thethiolgroupofcysteinewasprotectedusingp-methoxy benzyl chloride followed by its esterification in methanol and thionyl chloride to obtain S-p-methoxy benzyl cysteine methylesterhydrochlorideinabout80%yield(Scheme1).In itsIRspectrum,itshowedacharacteristicpeakat3412cm−1 correspondingtoN–Hstretching.Apeakat1745cm−1explains the presence ofester linkage.In theUVspectrum,it showed absorptionatλmax278nm.Inthe1HNMRspectrum,twomul- tipletswereobservedintherangeδ2.88–2.91andδ2.98–3.04 for diastereotopicprotonsHa andHb respectively,because of vicinalaswellasgeminalcoupling.Asingletwasobservedat δ 3.77 for methylene protonsof the benzylgroup as well as twosingletswereobservedatδ3.78andδ3.82whichaccounts for the presence of two methoxy groups. Adownfield triplet for thechiralprotonofcysteine(H-3)wasobservedatδ4.12 because of the presence of amino andesterfunctionalitiesin neighbouringpositions.Twodoubletsatδ6.88(J=8.7Hz)and δ7.28(J=8.7Hz)eachintegratingfortwoprotonswasassigned for aromaticprotonsandthiswasinaccordancewiththe1,4- disubstitutionofthephenylgroup.Inthe13CNMRspectrum, characteristicpeakscorrespondingtocarbonsofcysteinemoi- etywereobservedatδ31.62(C-4)andδ53.27(C-3).Thetwo methoxy carbons appeared atδ 52.66 andδ 55.97, while the carbonylcarbonwasobservedatδ169.52.
ThefinalstepstothetargetPNAbuildingblockcomprised theEDC-mediatedinstallationofthesuitablyprotectednucle- obasefollowedbysaponificationusing4Mlithiumhydroxidein methanol(Scheme3).Nucleobasesthymineandcytosinewere incorporatedviamethylenebridges.Sincethyminedoesnotcon- tain anysidechainwhichistobeprotectedsothymineacetic acidwasusedassuchbuttheexocyclicaminogroupofcytosine wasprotectedtopreventself-condensation.Theaminogroupof nucleobasecytosinewasprotectedusingthebenzyloxycarbonyl
NH O
O H
N O
O
S
OCH3
1 2
3 5 4
6 7 8 8
8 9
10 1′ 2′
3′ 4′
11
Ha Hb
7.268 1.16 1.00 0.51 1.77 2.66 0.53 0.84 0.99 1.58 0.84 1.16 6.83
7.240 7.212 6.863 6.835 5.019 4.133 4.109 4.085 4.034 3.837 3.798 3.727 3.688 3.510 3.436 3.386 3.364 3.345 3.317 3.300 3.163 2.754 2.734 2.715 2.696 2.673 2.627 2.583 2.044 1.781 1.448 1.282 1.259 1.235 1.151 1.130 0.000
8 7 6 5 4 3 2 1 0 ppm
Fig.2.1HNMRspectraof2-(Bocamino-ethylamino)-3-(4-methoxybenzylsulfanyl)-propionicacidmethylester(6).
Scheme1.SynthesisofS-p-Methoxybenzylcysteinemethylesterhydrochloride.
(Cbz)groupandthenintroductionofaceticacidwascarriedout forcouplingonbackbone(Scheme2).
Inthe1HNMRspectrumfor 2-[{2-(N4-Cbz-cytosin-1-yl)- acetyl}Boc-aminoethylamino]-3-(4-methoxybenzyl sulfanyl)- propionicacidmethylester(8)(Fig.3),acharacteristicsinglet fornine protonsforthree methylgroupsatδ 1.47 andcorre- spondingpeaksin13CNMR,atδ28.62alongwithδ79.29were duetothepresenceofthebocgroup.Themethylenegroupsof ethylenediaminebackbone(H-6andH-7)andprotonpresent atthe chiral centreof cysteine moiety(H-2) appeared inthe rangeδ3.02–3.38.Thetwodiastereotopicprotonsof cysteine HaandHbappearedasamultipletintherangeofδ4.59–4.88.
Themethyleneprotonsofthebenzyloxygroup(H-17)appeared atδ5.26 andamultipletfor thearomatic protonsof theCbz
groupalong withthearomatic protonsofcytosine(H-12and H-13) appeared in the range δ 7.31–7.51.That confirms the presence of Cbz-protectedcytosinemoiety. In the 13C NMR spectrum,peaksfortwomethylenecarbonsofethylenediamine moietywereobservedat δ39.82and49.24for C-6 andC-7, respectively.Characteristicpeaksforcytosinewereobservedat δ94.67(C-13)andδ151.35(C-12).Theamidecarbonyl(C-1) andestercarbonyl(C-1)appearedintherangeδ164.12–170.90.
InHRMS,the[M+H]•+peakwasobservedat684.1031havinga molecularformulaC33H41O9N5S(calculatedvalue:684.0625).
In the 1H NMR spectrum for 2-[{2-(Thymin-1-yl)- acetyl}boc-aminoethylamino]-3-(4-methoxybenzylsulfanyl)- propionic acid (9) (Fig. 4), a characteristic singlet for nine protons at δ 1.42 explainsthe presence of the bocgroup. A
Scheme2.Synthesisofbaseaceticacid.
Scheme3.Synthesisofthiol-modifiedPNAmonomercontainingpyrimidinenucleobases.
singlet for the methyl group at δ 1.81 for three protonswas assignedtothethyminemethylgroup.Thethymineringproton (H-10) appeared as multiplet along with aromatic protons (H-3)intherange δ7.25–7.29.Asmultipletsinthe rangeδ 3.30–3.50,themethylenegroupsofethylenediaminebackbone
(H-6andH-7)appearalongwiththeprotonpresentatthechiral centre of cysteine moiety (H-2). In the 13C NMR spectrum (Fig.5),peaksfortwomethylenecarbonsofethylenediamine moietywereobservedatδ39.76and49.01.Characteristicpeaks forthemethylgroupofthyminewasobservedatδ13.35.The
7.513 7.419 7.309 7.273 7.247 6.939 6.891 6.864 5.541 5.257 4.909 4.856 4.614 4.562 4.027 3.828 3.801 3.744 3.726 3.587 3.375 3.310 3.205 3.174 3.100 3.068 3.022 2.119 1.471 1.305 1.018 0.998 0.918 0.895 0.042
5.030.39
1.35
1.17
4.89
0.420.36
1.000.27
1.05
1.783.06
O 17 16 18 14 13
12
10 9
8
7 6 2
1 11
3
4 1″ 2″ 3″ 4″
5 10
10
15 2′
1′
O N N O O O
O N H
N Ha
Hb
OCH3 O
O S HN
8 7 6 5 4 3 2 1 0 ppm
Fig.3.1HNMRspectraof2-[{2-(N4-Cbz-cytosin-1-yl)-acetyl}Boc-aminoethylamino]-3-(4-methoxybenzylsulfanyl)-propionicacidmethylester(8).
NH O
O
N OH O
S
OCH3 O
N NH O
O
Ha Hb
1 2
3
4 7 6
8 9 9 9
1′ 2' 10 11
12 13
14
1″ 2″
3″
4″
5 15
7.292 2.61 1.95
8 7 6 5 4 3 2 1 0 ppm
0.40 1.76 0.76 5.59 4.16 1.38 7.30 3.00 9.00
7.264 7.254 6.875 6.846 4.832 4.777 4.712 4.658 4.286 3.766 3.706 3.500 3.482 3.429 3.406 3.385 3.360 3.297 3.166 3.150 3.119 3.100 3.062 3.015 2.057 2.050 2.042 2.035 2.028 1.808 1.418 1.387 0.183 –0.012 –0.210
Fig.4.1HNMRspectraof2-[{2-(Thymin-1-yl)-acetyl}boc-aminoethylamino]-3-(4-methoxybenzylsulfanyl)-propionicacid(9).
amidecarbonyl(C-1)andacidcarbonyl(C-1)wereassignedin therangeofδ164.86–168.40.TheidentityofthemodifiedPNA monomerswasconfirmedbyNMR,IRandmassspectrometry.
Combiningtheuseofthep-methoxybenzylgroupforthiol protectionandtheBocgroupforterminalNH2 hasled tothe simplepreparationofaduallabelledPNAmonomerwhichmay beusedasadiagnostictoolforbiomedicalstudies.Thesynthesis ofanewmercaptomethyl-modifiedPNAmonomerallowsthe applicationofnativechemicalligation-likefragmentcoupling
reactionswithoutdetrimenttobackbonegeometry.Itwasshown thattheDNAaffinityofthePNAligationproductsisashighas theaffinityofunmodifiedPNAobtainedbylinearsolid-phase synthesis.
While many PNA backbone modifications have shown the deleterious effecton PNA hybridization, it was observed that the addition of the mercaptomethyl group in PNA liga- tion products hardly influences the stability of a PNA/RNA duplex. This convergent approach may find applications in
168.40 164.86 159.77 156.86 153.46 151.91 142.62 131.29 131.04 114.85 114.70 109.83 79.37 61.39 55.54 49.19 49.01 39.76 36.36 31.24 30.61 30.35 30.25 30.09 29.98 29.95 29.84 29.76 29.74 29.72 29.70 13.35
180 160 140 120 100 80 60 40 20 0 ppm
Fig.5.13CNMRspectraof2-[{2-(Thymin-1-yl)-acetyl}boc-aminoethylamino]-3-(4-methoxybenzylsulfanyl)-propionicacid(9).
the synthesisandlabellingof difficult PNA-oligomersandin DNA-template-controlledligationreactions.Theapplicationof suitably protectedmodifiedPNA monomershasgivenaccess to a PNA oligomer containing a sulfhydryl group appropri- ateforpost–assemblyconjugation employingwellestablished conjugationmethods.
Ithasbeen observedthat theincorporationofasulfhydryl group in ethylene diamine moiety of PNA backbone (␥- position)didnotsubstantiallyaffectthehybridizationproperties with complementary RNA. However, the conjugation of the sulfhydrylgroupat␥-positionleadstothestericdestabilization of thePNA–DNA/RNAhybrid.Hence,inthispaperwe have focusedonthesynthesisoftwothiol–modified␣-monomers.
4. Conclusion
Orthogonallyprotectednovelthiol-modifiedPNAmonomers 9and10werepreparedcontainingthymineandcytosinenucle- obases.In thisreport, the thiol modification iscarried out in sidechain instead of ligationsite, i.e.mainethylenediamine backbone.Thesenovel␣-monomerswouldbeof relevancein studyinghybridizationpropertiesandavaluableassetforfuture conjugationofPNAswithavarietyofligandssuchasartificial RNA-nucleases for the sequence selectivedegradation of the targetRNA.
Conflictofinterest
Theauthorhasnoconflictsofinteresttodeclare.
Acknowledgement
Theauthorgratefullyacknowledgesthefinancialassistance fromtheScienceandEngineeringResearchBoard(DST),New Delhi, with Grant No. SB/FT/CS-057/2012. The author also acknowledges Departmentof Chemistry, University of Delhi foruseoffacilities.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.jart.2017.01.006.
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